Abstract：
DNA double-strand breaks (DSBs) are harmful to mammalian cells and a few of them can cause cell death. Accumulating DSBs in these cells to analyze their genomic distribution and their potential impact on chromatin structure is difficult. In this study, we used CRISPR to generate Ku80-/- human cells and arrested the cells in G1 phase to accumulate DSBs before conducting END-seq and Nanopore analysis. Our analysis revealed that DNA with high methylation level accumulates DSB hotspots in Ku80-/- human cells. Furthermore, we identified chromosome structural variants (SVs) using Nanopore sequencing and observed a higher number of SVs in Ku80-/- human cells. Based on our findings, we suggest that the high efficiency of Ku80 knockout in human HCT116 cells makes it a promising model for characterizing SVs in the context of 3D chromatin structure and studying the alternative-end joining (Alt-EJ) DSB repair pathway.
摘要：
DNA双链断裂（DSB）对哺乳动物细胞有害，其中一些会导致细胞死亡。在这些细胞中积累DSB以分析其基因组分布及其对染色质结构的潜在影响是困难的。在这项研究中,在进行END-seq和Nanopore分析之前，我们使用CRISPR生成Ku80-/-人细胞，并将细胞阻滞在G1期积累DSB。我们的分析显示，具有高甲基化水平的DNA在Ku80-/-人细胞中积累DSB热点。此外，我们使用Nanopore测序鉴定了染色体结构变体（SV），并在Ku80-/-人细胞中观察到更高数量的SV。根据我们的发现,我们认为,Ku80在人HCT116细胞中的高效敲除使其成为在3D染色质结构背景下表征SV和研究替代末端连接(Alt-EJ)DSB修复途径的有前途的模型.
